Derivatives of K5 polysaccharide having high anticoagulant activity

ABSTRACT

Derivatives of the K5 polysaccharide having anticoagulant activity higher than heparin, obtained by a process including the steps of reacting: with an organic base a solution of K5 polysaccharide N-deacetylated, N-sulfated and epimerized at least to an iduronic acid content of 50% treating with a sulfating agent to obtain the N-resulfation of the possible N-desulfated groups.

The present application is the national stage filing of and claimspriority to International Application No. PCT/EP97/02379, filed May 9,1997 and Italian Application Serial No. MI96A000956.

PRIOR ART

It is known that the product mainly used in anticoagulant therapy is theheparin obtained by extraction from animal organs. However theproduction of heparin from animal organs employs great amounts ofsolvents and chemical agents involving disposal and therefore potentialenvironmental pollution problems. Moreover the final product may containresidues of biological substances normally or exceptionally present inthe animal tissues as viruses or prions. O-sulfation processes carriedout on derivatives of the K5 polysaccharide (B. Casu et al.,Carbohydrate Letters, 1, 107-114 (1994)) are also known.

The publications by B. Casu et al. refer to O-sulfations carried out onK5 which result in products showing an anticoagulant strength lower thenthe commercial heparin.

This is also due to the fact that the entirety of uronic acids isrepresented by glucuronic acids. The glucuronic acid gives to thepolysaccharidic chain a lower flexibility towards the target proteinssuch as for example the antithrombin III and then a lower anticoagulantactivity (B. Casu, M. Petitou, M. Provasoli and P. Sinay (1988).Conformational flexibility: a new concept for explaining binding andbiological properties of iduronic acid containing glycosaminoglycans.Trends Biochem. Sci. 13, 221-225).

SUMMARY

Now a process for the preparation of new derivatives of the K5polysaccharide which allow to overcome the drawbacks of the prior arthas been found.

Said process includes the following steps:

a) the K5 polysaccharide is N-deacetilated;

b) the product obtained in the step a) is N-sulfated;

c) the product obtained in the step b) is epimerized to the achievementof at least 50% of iduronic acid with respect to the total of uronicacids, and it is characterized in that the product obtained in the stepc) is further treated according to the following steps:

d) the product obtained in the step c) is dissolved in water andpercolated through a column containing a cation exchange resin;

e) the solution obtained in the step d) is reacted with an organic base;

f) the solution obtained in the step e) is freeze-dried and the obtainedproduct is redissolved in an organic solvent and treated with asulfating agent to obtain the O-sulfation;

g) the product obtained in the step f) is precipitated, redissolved indistilled water and dialyzed against distilled water;

h) If it is necessary, the product obtained in the step g) is treatedwith a sulfating agent in order to obtain the N-resulfation of the incase N-desulfated groups.

Optionally the product obtained after the step h) is depolymerized bycontrolled nitrous acid degradation according to known technology (i.e.U.S. Pat. No. 5,019,649).

The products according to the present invention have, beside newcharacteristics, a high anticoagulant activity, greater than theanticoagulant activity of the heparin obtained by extraction from animaltissues.

FIG. 1 represents the data relative to the affinity of heparin forantithrombin III.

FIG. 2 represents the data relative to the affinity of the derivative ofExample 1 for antithrombin III.

FIG. 3 represents the data relative to the affinity of the derivative ofExample 1 for antithrombin III.

FIG. 4 represents the data relative to the affinity of the derivative ofExample 3 for antithrombin III.

DETAILED DESCRIPTION OF THE INVENTION

The characteristics and the advantages of the derivatives of the K5polysaccharide and the relative preparation process according to thepresent invention will be mostly pointed out during the followingdetailed description. The starting material for the achievement of saidderivatives is the K5 polysaccharide obtained from E. Coli as describedby M. Manzoni, S. Bergomi and V. Cavazzoni (Journal of Bioactive andCompatible Polymers. Vol. VIII, July 1993, 251-257). The K5polysaccharide is first of all treated as in the following steps:

a) the K5 polysaccharide is N-deacetilated;

b) the product obtained in the step a) is N-sulfated in order to obtainN-sulfated K5 from 25 to 100%;

c) the product obtained in the step b) is epimerized with D-glucuronylL-iduronyl C5 epimerase extracted from bovine liver in order to obtain aproduct having a L-iduronic acid content from 50 to 90% with respect tothe total of the uronic acids.

The N-deacetilation of the step a) is carried out by treatment with ahydrazine and hydrazine sulfate mixture or in an alkaline environmentwith sodium hydroxide or potassium hydroxide. Subsequently theN-sulfation of the step b) is carried out by treatment withtriethylamine-sulfur trioxide or with trimethylamine-sulfur trioxide.The N-deacetilation and N-sulfation reactions are carried out accordingto the known techniques, for example according to the Patent WO92/17507.

The N-sulfated product is then submitted to the epimerization reactionof the step c) in order to convert the glucuronic acid in iduronic acid.The epimerization is carried out present theD-glucuronyl-L-iduronyl-C5-epimerase enzyme (hereinafter simply denotedwith C5-epimerase) extracted from bovine liver and purified by themethod described by A. Malmstrom in J. B. C. 255, 3878-3883 (1980). Thereaction medium is a buffer solution at pH 7.4 for example consisting ofHEPES 0.04 M or TRIS 0.05 M, potassium chloride, EDTA and TRITON X-100and added with one or more additives selected from the group consistingof glycol, glycerol, polyvinylpyrrolidone, particularlypolyvinylpyrrolidone having a molecular weight from 15,000 to 90,000,glycol and lecithin in such an amount to increase the viscosity of thebuffer solution to values ranging from 1.1 to 3 centistokes. Inparticular the reaction medium is prepared starting from a suitablebuffer solution having pH 7.4; such as for example HEPES 0.04 M, KCl 0.4M and EDTA 0.06 M and to 25 ml of this solution from 100 to 1000 μl ofTRITON X-100, from 0.5 ml to 60 ml of additive and distilled water to100 ml are added. The polysaccharide to submit to epimerization is addedto said reaction medium in amounts from 5 to 1000 mg per 100 mlobtaining the solution A. The C-5 epimerase is 20 dissolved separatelyin the same reaction medium above mentioned in amounts from 21 to 2000μg per 100 ml obtaining the solution B. The solution B is added to thesolution A in such a proportion to obtain a C-5 epimerase content from1.5 to 15,000 μg per 100 ml of mixture to submit to epimerization.

The mixture is homogenized by stirring and heated at a temperatureranging from 30 to 40° C. in a constant-temperature chamber for a timeranging from 90 minutes to 15 hours.

The reaction in stopped heating the mixture at 100° C. for 5 minutes.The product is purified through a DEAE-Sephacel column using (NH₄)HCO₃or NaCl 0.05 M as buffer and eluting the product with buffer (NH₄)HCO₃or NaCl 2 M.

The gathered fractions are desalted by Sephadex G-15 column, thefraction containing the product is freeze-dried and the product isanalyzed by 1H-NMR. From the 1H-NMR spectrum the D-glucuronic acid andthe L-iduronic acid content is computed.

The obtained product may be redissolved in the solution A and treatedagain with the solution B obtaining, with further epimerizationtreatments, an increase of the L-iduronic acid content.

The product obtained from the step c) as described above is furthertreated as described in the following steps, which characterize thepresent invention:

d) the product obtained in the step c) is dissolved in water andpercolated through a column containing a cation exchange resin such asfor example Amberlite IR 120 H⁺ (Rohm and Haas) which is subsequentlywashed with distilled water.

The pH of the obtained solution ranges from 0.5 to 1.5;

e) the solution obtained in the step d) is treated with an organic basepreferably selected from the group consisting of trimethylamine,triethylamine and tributylamine, dissolved in an organic solvent such asfor example alcohol. The organic base amount added is such to obtain asolution pH ranging from 6.5 to 7.0. The organic base excess is removedby treatment with diethyl ether;

f) the solution obtained in the step e) is freeze-dried and the obtainedproduct is redissolved in an organic solvent at room temperature andtreated with a sulfating agent at a temperature ranging from -5 to 60°C. for a time period ranging from 10 to 24 hours, in order to obtain theO-sulfation.

Said organic solvent is preferably the anhydrous dimethylformamide andsaid sulfating agent is preferably selected from the group consisting ofpyridine sulphur trioxide, trimethylamine sulphur trioxide,triethylamine sulphur trioxide, tripropylamine sulphur trioxide andtributyl amine sulphur trioxide;

g) the solution obtained in the step f) is diluted with an equal volumeof water, a solution of NaOH at 4% is added to reach a pH equal to 9 andthe product is precipitated by addition of 4 volumes of alcoholsaturated by sodium acetate and maintaining the temperature from 3 to 5°C. for 10-15 hours. The obtained precipitate is dissolved in distilledwater and dialyzed against distilled water in a 1,000 cut-off dialysismembrane for 3 days with extra-dialysis change every day;

h) if it is necessary, the solution obtained in the step g) is addedwith sodium bicarbonate to pH 9, it is heated at a temperature ragingfrom 50 to 60° C. and a sulfating agent selected from the group pointedout in the step f) is added in order to obtain the N-resulfation of thegroups in case desulfated during the treatment. This reaction is carriedout under stirring for a period of time ranging from 5 to 10 hours, at atemperature ranging from 50 to 60° C.

At the end the solution is desalted by a 3500 D dialysis againstdecreasing solutions of NaCl for S days and the product is freeze-dried.

Optionally the product obtained after the step h) is depolymerized bycontrolled nitrous acid degradation according to known technology (i.e.U.S. Pat. No. 5,019,649).

K5 polysaccharides derivatives having new characteristics and ananticoagulant activity greater than the heparin one obtained by theextraction from animal tissues are obtained by the described process.The derivatives according to the present invention contain from 40 to100% of chains affine for the Antithrombin III, computed according tothe method described by M. Hook et al. (FEBS letters, 66, 1976, 90-93),while the heparin contains only 30% of chains affine for theAntithrombin III and this explains its greater anticoagulant activity.In the following Table 1 the values of the chemical analysis and theanticoagulant activity in vitro of the derivatives according to theinvention (A) in comparison with the commercial heparin (B) arereported.

                  TABLE 1                                                         ______________________________________                                                       (A)     (B)                                                    ______________________________________                                        Sulfates/carboxyls ratio                                                                       2.2-2.5   1.9-2.4                                            N-sulfates content                                                                              70%-100% 86%-91%                                            6-O-sulfates content                                                                           70%-90%   64%-89%                                            2-O-sulfates content                                                                           50%-60%   71%-78%                                            3-O-sulfates content                                                                            5%-10%   0.5%-2.0%                                          Chains affine for                                                                               40%-100% 28-35                                              the Antithrombin III                                                          Anti-Xa          500-600   145-197                                            APTT             250-320   145-187                                            ______________________________________                                    

The sulfates/carboxyls ratio has been determined by the conductimetricmethod according to B. Casu et al. (Carbohyd. Res. 39.168 (1975)) whilethe sulfates distribution has been determined by nuclear magneticresonance according to B. Casu et al. (Arzneim. Forsch./Drug Res. 33(I),1, 135-142-1983).

The 3-O-sulfates content has been determined by the method described byB. Casu et al. (Biochem J. 197, 1981, 599-609).

The anticoagulant activity has been measured as APTT according to L.Andersson et al. (Thrombosis Res. 9, 575-1976), and as Anti Xa accordingto D. P. Thomas et al. (Thrombosis and Haemostasis 45, 214-1981).

Thanks to their characteristics the derivatives according to the presentinvention may be used for the preparation of pharmaceutical compositionssuitable to the anticoagulant treatment in the human therapy.

Said compositions contain efficacious amounts of said derivatives incombination with pharmacologically acceptable excipients or diluents.The posology for the human therapy is from 30 to 200 mg per day.

The derivatives according to the present invention also exhibit withrespect to heparin the great advantage to be viruses and prions free andthe production process has the advantage not to give pollutingeffluents.

EXAMPLE 1

10 mg of 100% N-sulfated and 70% epimerized (that is containing 70% ofiduronic acid with respect to the uronic acids total) K5 have beendissolved in 2 ml of water and put into an Amberlite IR 120H⁺ column atroom temperature. The column has been washed with 10 ml of water. Theeluate plus the washing liquid had a pH equal to 1.5. The solution hasbeen added with tributylamine to pH 5.5 using a solution oftributylamine at 10% in ethanol. The excess of tributylamine not boundto the polysaccharide has been removed by treatment with diethyl ether.The solution has been finally freeze-dried.

Then the product has been redissolved in 3.2 ml of anhydrousdimethylformamide at room temperature and 3 ml of anhydrousdimethylformamide containing 0.153 g of piridine-sulphur trioxide havebeen added. The obtained solution has been kept at room temperature for6 hours and then diluted with an equal volume of water. The pH has beenfinally set to 9 with NaOH at 4% and the product has been precipitatedwith 4 volumes of ethanol saturated with sodium acetate keeping thesolution at 4° C. overnight. The obtained precipitate has been dissolvedin 10 ml of distilled water and dialyzed against distilled water in a1,000 cut-off dialysis membrane for 3 days with extra-dialysis changeevery day.

The obtained sample has been submitted to N-resulfation. The pH has beenset to 9 with the addition of solid sodium bicarbonate, the temperaturehas been raised to 55° C. and 6.5 ml of trimethylamine-sulphur trioxidehave been added under stirring. The solution has been kept at 55° C. for1 hour, further 6.5 ml of trimethylamine-sulphur trioxide have then beenadded and the reaction carried on for additional 5 hours. The sample hasbeen desalted by 3500 D dialysis against solutions having decreasingNaCl concentration for 5 days (0.5 M the first day, 0.2 M the secondday, 0.1 M the third day and water the fourth and the fifth day). Theproduct has been finally freeze-dried.

The obtained product exhibits a sulfates/carboxyls ratio equal to 2.5,100% N-sulfates content, 80% 6-O-sulfates content, 60% 2-O sulfates, 10%3-O sulfates, a fraction affine to the AT III equal to 100% and thefollowing in vitro anticoagulant activities:

    ______________________________________                                               Anti-Xa       600 U/mg                                                        APTT          310 U/mg                                                 ______________________________________                                    

EXAMPLE 2

10 mg of 90% N-sulfated and 60% epimerized K5 have been treated as inthe Example 1 with the difference that, after the N-resulfation, theproduct has been treated at a temperature equal to 40° C. with 10 ml ofanhydrous dimethylformamide containing 0.51 g of pyridine-sulfurtrioxide added under stirring.

After 2 hours further 10 ml of anhydrous dimethylformamide containing0.51 g of pyridine-sulfur trioxide have been added and the reaction hasbeen continued for additional 10 hours.

The product has been desalted as in the Example 1.

The obtained product exhibits a sulfates/carboxyls ratio equal to 2.4,90% N-sulfates content, 100% 6-O sulfates content, 40% 2-O sulfates, 7%3-O sulfates, a fraction affine to the AT III equal to 85% and thefollowing in vitro anticoagulant activities:

    ______________________________________                                               Anti-Xa       550 U/mg                                                        APTT          290 U/mg                                                 ______________________________________                                    

EXAMPLE 3

10 mg of 80% N-sulfated and 55% epimerized K5 have been treated as inthe Example 1 with the difference that, after the N-resulfation, theproduct has been treated at a temperature equal to 65° C. with 10 ml ofanhydrous dimethylformamide containing 0.51 g of pyridine-sulfurtrioxide added under stirring.

After 2 hours further 10 ml of anhydrous dimethylformamide containing0.51 g of pyridine-sulfur trioxide have then been added and the reactionhas been continued for additional 5 hours.

The product has been desalted as in the Example 1.

The obtained product exhibits a sulfates/carboxyls ratio equal to 2.5,80% N-sulfates content, 100% 6-O sulfates content, 65% 2-O sulfates, 5%3-O sulfates, a fraction affine to the AT III equal to 70% and thefollowing in vitro anticoagulant activities:

    ______________________________________                                               Anti-Xa       450 U/mg                                                        APTT          270 U/mg                                                 ______________________________________                                    

In the Table 2 the data relative to the characteristics of the productsof the Examples have been summarized, from which one may noticeparticularly that the anticoagulant activity decreases with the decreaseof the iduronic acid content.

EXAMPLE 4

10 mg of the product obtained in the Example 3 are dissolved in 10 ml ofdistilled water and added with 0.34 mg of sodium nitrite.

Immediately the pH is brought to 2.5 with hydrochloric acid 0.01 N.After 40 minutes the solution is neutralised with sodium hydroxide andthe compound is recovered by precipitation with 3 volumes of ethanol anddried in a vacuum oven.

The compound obtained shows a sulphate/carboxyl ration of 2.2,N-sulphate content of 70%, 6-O sulphate content of 65%, 2-O sulphatecontent of 60%, 3-O sulphate content of 4%, an ATIII high activityfraction of 40% and the following in vitro anticoagulant activities:

    ______________________________________                                               Anti-Xa       200 U/mg                                                        APTT           70 U/mg                                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                   Example 1                                                                            Example 2                                                                              Example 3                                                                              Example 4                                 ______________________________________                                        Sulfates/carboxyls ratio                                                                   2.5      2.3      2.4    2.2                                     Mean molecular weight                                                                      14,000   14,000   14,000 5,000                                   N-sulfates   100%     90%      80%    70%                                     6-O sulfates 80%      90%      70%    65%                                     2-O sulfates 60%      50%      60%    60%                                     3-O sulfates 10%       7%       5%     4%                                     Iduronic Acid                                                                              70%      60%      55%    55%                                     Fraction having high                                                                       100%     85%      70%    40%                                     affinity for AT III                                                           Anti-Xa      600 U/mg 550 U/mg 500 U/mg                                                                             200 U/mg                                APTT         310 U/mg 290 U/mg 250 U/mg                                                                              70 U/mg                                ______________________________________                                    

In the FIGS. 2, 3, and 4 the data relative to the affinity for theantithrombin III for the products of the Examples 1, 2 and 3 arereported, while in the FIG. 1 are reported, by comparison, the same datafor the heparin.

In said Figures:

mAbs 530 nm means milliabsorbance determined at 530 nm;

LA means fraction having low affinity for the antithrombin III;

HA means fraction having high affinity for the antithrombin III;

These data confirm what has been reported as commentary of the Table 2.

What is claimed is:
 1. Process for the preparation of derivatives of the K5 polysaccharide having anticoagulant activity higher than heparin, wherein:a) the K5 polysaccharide, N-deacetylated, N-sulfated and epimerized at least to a iduronic acid content equal to 50%, with respect to the total of uronic acids, is dissolved in water and percolated through a column containing a cation exchange resin; b) the solution obtained in the step a) is reacted with an organic base; c) the-solution of the step b) is freeze-dried and the obtained product is redissolved in an organic solvent and treated with a sulfating agent to obtain the O-sulfation; d) the product obtained in the step c) is precipitated, redissolved in distilled water and dialyzed against distilled water; e) the product obtained in step d) is if necessary treated with a sulfating agent in order to obtain the N-resulfation of any N-desulfated groups.
 2. Process as claimed in claim 1, characterized in that the solution obtained in step a) has a pH ranging from 0.5 to 1.5.
 3. Process as claimed in claim 1, characterized in that said organic base used in step b) is selected from the group consisting of trimethylamine, triethylamine and tributylamine.
 4. Process as claimed in claim 1, characterized in that the amount of organic base used in step b) is such to obtain a solution pH ranging from 6.5 to 7.0.
 5. Process as claimed in claim 1, characterized in that said organic solvent used in step c) consists of anhydrous dimethylformamide.
 6. Process as claimed in claim 1, characterized in that said sulfating agent used in step c) is selected from the group consisting of pyridine-sulfur trioxide, trimethylamine-sulfur trioxide, triethylamine-sulfur trioxide, tripropylamine-sulfur trioxide and tributylamine-sulfur trioxide.
 7. Process as claimed in claim 1, characterized in that said treatment with a sulfating agent of step c) is carried out at a temperature ranging from -5 to 60° C. for a time period ranging from 10 to 24 hours.
 8. Process as claimed in claim 1, characterized in that said precipitation of step d) is carried out by diluting the solution obtained in step c) with an equal volume of water, adjusting the pH to 9, adding 4 volumes of alcohol; saturated with sodium acetate and keeping the temperature in the range of 3 to 5° C. for a time period ranging from 10 to 15 hours.
 9. Process as claimed in claim 1, characterized in that said sulfating agent used in step e) is selected from the group consisting of pyridine-sulfur trioxide, trimethyl-sulfur trioxide, triethylamine-sulfur trioxide, tripropylamine-sulfur trioxide and tributylamine-sulfur trioxide.
 10. Process as claimed in claim 2, characterized in that said N-resulfation reaction of step e) is carried out at pH 0, at a temperature ranging from 50 to 60° C. for a time period ranging from 5 to 10 hours.
 11. Derivatives of the K5 polysaccharide epimerized at least to 50% of iduronic acid with respect to the total of uronic acid, having:

    ______________________________________                                         sulfate/carboxyl ratio  2.2-2.5                                                N-sulfates content      70-100%                                                6-O-sulfates content    70-90%                                                 2-O-sulfates content    50-60%                                                 3-O-sulfates content    5-10%                                                  Chains affine for the   40-100%                                                Antithrombin III                                                               Anti - Xa              500-600                                                 APTT                   250-320.                                                ______________________________________                                    


12. Pharmaceutical compositions suitable for anticoagulant treatment of humans, said compositions consisting of effective amounts of derivatives of the K5 polysaccharide as claimed in claim 11, in combination with pharmacologically acceptable excipients or diluents.
 13. Therapeutic method for anticoagulant treatment of humans, said method consisting in the administration of a derivative of the K5 polysaccharide as claimed in claim 11, in amounts of 30 to 200 mg per day.
 14. Process as defined in claim 1 wherein the product obtained in step e) is depolymerized by controlled nitrous oxide degradation. 